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United States Patent |
5,156,019
|
McCormick
|
October 20, 1992
|
Frozen tissue sectioning apparatus and method
Abstract
Disclosed is an apparatus for slicing tissue sections from a frozen
specimen for microscopic analysis or the like without allowing tissue
debris or dust to become airborne and thereby transport bacteria or
viruses from one specimen to other specimens or to an operator. This is
achieved by slicing the specimen while submersed in a subzero dielectric
liquid and passing a conveyor, such as a statically charged web or film,
adjacent the point of sectioning so that the tissue sections and
associated tissue debris generated therewith are attracted to the web and
adhere thereto. The conveyor transports the tissue sections, and
associated debris adhering thereto, through a sterilizer and staining
tanks whereafter the tissue is transferred to a slide for microscopic
examination.
Inventors:
|
McCormick; James B. (6755 Longmeadow Dr., Lincolnwood, IL 60646)
|
Appl. No.:
|
615493 |
Filed:
|
November 19, 1990 |
Current U.S. Class: |
62/320; 83/915.5 |
Intern'l Class: |
F25C 005/02 |
Field of Search: |
62/320
83/915.5
|
References Cited
U.S. Patent Documents
3204424 | Sep., 1965 | McCormick | 62/320.
|
3233965 | Feb., 1966 | McCormick | 83/915.
|
3272348 | Sep., 1966 | Metz | 83/915.
|
3462969 | Aug., 1969 | Grasenick et al. | 62/320.
|
3491638 | Jan., 1970 | Idlis | 83/915.
|
3664412 | May., 1972 | Zerkle | 62/320.
|
4548051 | Oct., 1985 | Moessner | 62/320.
|
4979376 | Dec., 1990 | Biehl et al. | 83/915.
|
Foreign Patent Documents |
2141129 | Mar., 1973 | DE | 83/915.
|
426164 | Oct., 1974 | SU | 83/915.
|
Primary Examiner: Bennet; Henry A.
Assistant Examiner: Kilner; Christopher B.
Attorney, Agent or Firm: Fitch, Even, Tabin & Flannery
Claims
What is claimed is:
1. A cryogenic microtome apparatus for sectioning frozen tissue specimens,
comprising:
container means adapted to contain a dielectric fluid;
microtome means submersed in the dielectric fluid for sectioning a tissue
section from a frozen tissue specimen;
a conveyor means for conveying the tissue section from said container
means;
means for mounting said conveyor means immediately adjacent said microtome
means; and
means for causing adhesion between a tissue section and associated debris
and said conveyor means.
2. A microtome apparatus in accordance with claim 1 wherein said apparatus
includes means for sterilizing the tissue section and associated debris
upon transportation from the dielectric fluid while maintaining their
chemistry and antigenicity.
3. A microtome apparatus in accordance with claim 2 wherein, after
undergoing sterilization, tissue sections and their associated tissue
particles are transported to a mounting station for analysis.
4. A microtome apparatus in accordance with claim 1 wherein the apparatus
includes means for synchronizing the movement of said conveyor with the
rate of tissue section generation.
5. A microtome apparatus in accordance with claim 4 wherein said means for
synchronizing the movement of said conveyor with the rate of tissue
section generation includes a logic-controlled stepping motor which
advances said film a given distance for each tissue section generated.
6. A microtome apparatus in accordance with claim 1 wherein said means for
sectioning organic tissue includes a stationary tissue slicing means and
further includes a moving tissue block adjacent each other, said organic
tissue being mounted on said block.
7. A microtome apparatus in accordance with claim 6 wherein an oscillating
drive means oscillates the tissue block linearly with respect to said
tissue slicing means.
8. A microtome apparatus in accordance with claim 1 wherein said conveyor
means includes a dielectric film.
9. A microtome apparatus in accordance with claim 8 wherein said dielectric
film includes an elongated film web of plastic.
10. A microtome apparatus in accordance with claim 9 wherein said film and
section have opposite electrical surface charges imposed thereon.
11. A microtome apparatus in accordance with claim 1 wherein said conveyor
means includes a continuous, traveling web of dielectric solid passing
adjacent said microtome means at a point on its path of travel.
12. A microtome apparatus in accordance with claim 2 wherein the apparatus
includes a series of tanks for receiving staining and fixing agent and
means for passing said conveyor therethrough.
13. A microtome apparatus in accordance with claim 2 wherein said means for
sterilizing said tissue sections adhering to the dielectric solid includes
an oxidizing lamp for oxidizing tissue passing in close proximity to the
oxidizing lamp.
14. A microtome apparatus in accordance with claim 1 including a means for
transfer attaching the tissue section from the plastic web to a glass
slide with a U.V. polymerized resin mounting material.
15. A microtome apparatus in accordance with claim 3 including gas- and
liquid-phase processing means for processing said tissue prior to said
transportation thereof to a mounting station for analysis.
16. A microtome apparatus in accordance with claim 1 wherein said
dielectric fluid is maintained between approximately -40.degree. C. and
0.degree. C., preferably at -20.degree. C.
17. A method for the preparation of tissue sections comprising:
sectioning a tissue specimen at a sectioning station to provide a tissue
section and associated debris;
adhering said section and debris to the surface of a conveyor;
conveying said section and associated debris to a mounting station remote
from said sectioning station;
removing said tissue section from said conveyor at said mounting station;
and
mounting said tissue to provide a microscopic slide.
18. A method in accordance with claim 17 wherein the sectioning station is
immersed in a dielectric fluid.
19. A method in accordance with claim 17 wherein the tissue specimen is
frozen.
20. A method in accordance with claim 18 wherein the dielectric fluid is at
a temperature of between about -10.degree. C. and about -40.degree. C.
21. A method for sectioning organic tissue under cryogenic conditions,
comprising:
submersing a frozen tissue specimen to be sectioned in a subzero dielectric
liquid means;
passing a dielectric film in proximity with said frozen tissue specimen;
imposing an electrical surface charge to one of said dielectric film and
said organic tissue such that as tissue is sectioned, the tissue section
and its associated tissue particles electrostatically adhere to the
dielectric film passing in proximity with the tissue;
sectioning said tissue by sectioning means while the tissue is submersed in
said dielectric liquid; and
transporting the sectioned tissue and tissue particles from the dielectric
liquid.
22. A method in accordance with claim 21 including the step of sterilizing
the tissue sections while they are electrostatically adhered to the film.
23. A method in accordance with claim 22 including the step of transporting
the sterilized tissue sections to a mounting station for viewing.
24. A method in accordance with claim 21 including the step of passing
tissue sections through gas- and liquid-phase processing means.
25. A method in accordance with claim 21 including the step of
synchronizing the movement of said dielectric solid with the rate of
tissue section generation.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to an apparatus and method for sectioning frozen
organic tissue samples under cryogenic conditions.
2. Description of Related Art
It is known in the prior art to freeze tissue samples prior to sectioning
in order to allow for greater precision in tissue sectioning. For
instance, the tissue sample can be frozen to a particular shape prior to
sectioning in order to produce tissue sections of a uniform, desired
cross-section. Various apparatus, such as that disclosed in U.S. Pat. Nos.
3,204,424 and 3,213,379, have been developed for freezing tissue specimens
in preparation for sectioning in a microtome. It is necessary, therefore,
to employ a cryostat microtome which maintains the tissue specimen in a
frozen state during sectioning. Current cryostat microtomes, such as that
disclosed in U.S. Pat. No. 4,548,051, operate in a cold air environment
wherein an enclosure maintains a refrigerated cold air environment within
which the microtome is housed. Such cold air microtomes suffer from
several shortcomings, as explained below.
A microtome is a piece of precision equipment for making very thin sections
(1 to 15 .mu.m) from tissue. A microtome may have to section large numbers
of tissue samples daily. These tissue samples may contain countless
viruses and bacteria. Due to the fact that slicing organic tissue thinly
necessarily results in the simultaneous generation of some amount of
tissue debris or "dust," slicing of large numbers of tissue samples
necessarily results in generation of large amounts of tissue debris.
Accordingly, conventional cold-air cryostat microtome arrangements have
been found to result in large amounts of tissue debris becoming airborne.
This tissue debris contains whatever viruses and bacteria that were
present in the original tissue specimen. Thus, one problem associated with
prior cryostat microtomes is that some originally pure tissue specimens
are contaminated by airborne tissue particles of other specimens, thereby
greatly reducing the accuracy of analyses and/or examinations of such
tissue sections.
Also, due to the precision required in producing such thinly sliced tissue
sections, close operator supervision is necessary. Thus, in using
conventional cryostat microtomes, operators are required to be exposed to
the airborne tissue particles generated upon tissue sectioning. There is,
accordingly, a great fear of operator contamination from the viruses and
bacteria present in these airborne particles. This fear has been
particularly accentuated in recent years by increased cases of hepatitis
and AIDS. Thus, operators are currently reluctant to operate conventional
microtomes or work near them.
Current microtomes generate significant amounts of tissue debris during
sectioning. Thus, there is a need for the ability, in such microtomes, to
be able to section large quantities of tissue without allowing the tissue
debris generated therewith to become airborne.
SUMMARY OF THE INVENTION
In accordance with the present invention, tissue sections are sliced from a
specimen without tissue dust, debris, or other tissue particles,
collectively referred to herein as debris, becoming airborne and thereby
transporting bacteria such as tuberculosis and fungi, or viruses such as
AIDS or hepatitis. Preferably, this is achieved by providing a vessel or
other suitable container which is filled with a dielectric fluid
maintained at a subzero temperature. A microtome or other suitable
sectioning device is submersed in the dielectric fluid, slicing the
specimen being sectioned while submersed in a dielectric liquid and a
tissue conveyor, generally in the form of a web or film, is passed closely
adjacent to the microtome and an attractive charge is created between the
specimen and the conveyor that causes the removed tissue section and any
tissue debris generated to be attracted to and adhered to the conveyor.
The conveyor, with the tissue section and associated debris adhered
thereto, is transported through a sterilizer where the tissue section and
debris are sterilized, preferably either by means of radiation or by means
of a strong oxidizing lamp generating ozone, while keeping the chemistry
and antigenicity of the tissue section intact. The sterilized tissue
section and tissue particles are subsequently transported through staining
tanks whereat routine or special stains are applied, and finally to a
mounting station at which the sections are removed from the conveyor and
are mounted for later analysis, usually by potting the sample in a
methacrylate or other suitable monomer that is polymerized by exposure to
ultraviolet light. Conventional microscopic examination on a glass slide
may then be made.
BRIEF DESCRIPTION OF THE DRAWINGS
This invention will be more fully understood, and further objects and
advantages thereof will become apparent in the following detailed
description of preferred embodiments of the invention illustrated in the
accompanying drawings, wherein like elements are referenced alike.
FIG. 1 is an illustration of an embodiment of a cyrostat microtome
apparatus constructed in accordance with the present invention; and
FIG. 2 is an enlarged perspective view of the sprocketed wheels employed in
accordance with the embodiment of FIG. 1.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
FIG. 1 shows a preferred embodiment of a cryostat microtome apparatus
constructed according to the principles of the present invention. The
overall apparatus is generally designated at 10, while the microtome
itself, which performs the tissue sectioning, is generally designated at
12.
The microtome 12 is preferably of the kind already known in the art,
wherein a tissue specimen 14 is mounted to a tissue block 16 which
oscillates beneath a stationary blade 18 to section the tissue specimen 14
thereon. As shown in FIG. 1, in accordance with the present invention, the
microtome 12 is housed within an insulated tank 20 and submersed in a
dielectric oil 22. Preferably, the dielectric oil 22 is at a subzero
temperature so that a frozen tissue specimen remains frozen during
sectioning.
A conveyor 24 is mounted for passage immediately adjacent to the stationary
blade 18, and an attractive charge is established between the specimen and
the conveyor 24 so that tissue section 26, and the tissue debris generated
therewith when severed from the specimen, will be attracted to and adhered
to the conveyor 24, generally by electrostatic attraction. The advancement
of the conveyor 24 is synchronized with the rate of tissue section
generation so that each tissue section 26, with its associated tissue
debris, occupies a separate space on the conveyor 24 separated from other
tissue sections and their associated debris.
The tissue sections 26, electrostatically adhering to the conveyor 24, are
transported out of the subzero environment of the insulated tank 20 upon
advancement of the conveyor 24, and are immediately sterilized in a
suitable sterilizer 56 to sterilize any bacteria or viruses present. The
sterilized tissue sections 26 may subsequently pass through additional
processing sections, as is conventional in the art, whereafter they are
finally transported to a mounting station 60 whereat sections are mounted
for ultraviolet or microscopic examination.
This arrangement allows large quantities of tissue to be rapidly sectioned
without allowing the tissue debris generated therewith to become airborne
and preventing the debris from one section to come into contact with, and
thereby contaminating, another tissue section. The present invention is
particularly well-suited for making series sections, which are a series of
sections taken from the specimen in question, without gaps or intervals
between the sections produced.
With reference to FIG. 1, a conventional microtome 12 is employed to
section a frozen tissue specimen 14. The microtome 12 is mounted to the
bottom of an insulated tank 20, which tank contains dielectric oil 22 at a
level sufficient to completely submerse the microtome 12 therein. The oil
22 is maintained generally between -10.degree. C. and -40.degree. C., and
preferably at approximately -20.degree. C., by a suitable refrigeration
unit, not shown, so that the tissue specimen 14 and tissue sections 26
remain frozen while within the insulated tank. Any dielectric oil that is
sufficiently fluid at these temperatures, may be employed. The insulation
in the tank 20 is provided to assist in maintaining the subzero
temperature therein.
As stated above, the microtome 12 includes a tissue block 16 upon which a
tissue specimen 14 is mounted, and which oscillates beneath a stationary
blade 18. The tissue block 16 is preferably comprised of a stainless steel
disc 28 upon which the specimen is mounted and a depending mandrel 30
which supports the disk. The tissue block 16 component of the microtome is
removably attached to the microtome 12 by insertion of the mandrel 30
therein. A standard tool changer can be employed to insert successive
tissue blocks 16 with specimens 14 mounted thereon in the microtome for
sectioning, and to remove the tissue blocks 16 from the microtome 12 upon
completion of the sectioning.
With the tissue block 16 inserted in the microtome 12, sectioning is
accomplished by employing a linear step motor 32 to oscillate the tissue
block 16 laterally beneath the stationary blade 18 slicing the tissue
specimen 14 with each lateral oscillation. Also, a micrometer feed step
motor 34 is employed to adjust the transverse position of the tissue block
16 relative to stationary blade 18 between successive lateral
oscillations. That is, the thickness of the generated tissue sections 26
can be varied, as desired, by adjusting the micrometer feed step motor 34
to move the tissue block 16 transversely either a large or small amount
between successive slicings. Accordingly, tissue specimens 14 are
sectioned in a rapid, automated process while retained in a frozen state
during sectioning.
With each such tissue sectioning, there is usually generated a
corresponding quantity of tissue debris. To prevent such tissue debris
from becoming airborne or contaminating other tissue sections, a conveyor
24, having a surface charge thereon, is passed in close proximity to the
microtome 12 to cause the tissue sections 26, and associated tissue debris
generated therewith, to electrostatically adhere to the conveyor 24.
The conveyor 24 can be advanced by any suitable means. In one embodiment,
as seen in FIG. 2, to provide for advancement of the conveyor which is in
the form of a flexible web, the conveyor 24 is fenestrated adjacent both
its lateral sides 36. That is, sprocket holes 38 are provided along the
length of the lateral sides 36 of the conveyor 24, which are evenly spaced
from one another and which receive sprockets 42 therein. As seen in FIG.
1, the conveyor 24 is preferably passed around several sprocketed wheels
40. The registration of the sprockets 42 and the sprocket holes 38
maintains accurate alignment of the conveyor 24. These sprocketed wheels
40 are located at opposing ends of axles 45. As shown in FIG. 2, this
sprocket arrangement allows sufficient clearance between the conveyor 24
and axles 45 so that the tissue sections 26, adhering to the underside of
the conveyor 24, do not come in contact with the axles 45 when passing
thereover.
In the embodiment of FIG. 1, the conveyor 24 is driven by a drive roller 46
having sprocketed drive wheels 48 at its opposing ends. The drive roller
46 is, in turn, driven by a linear motor 50 which is provided with logic
control operating in conjunction with feedback from the micrometer to
advance the conveyor 24 at the rate of tissue section generation. Thus, as
seen in FIG. 1, the conveyor 24 is initially wound about a supply reel 44,
and is ultimately received upon a take-up reel 52, after passing around
several sprocketed wheels 40 under the action of the drive roller 46. That
is, the conveyor 24 makes a single pass from the supply reel 44 to the
take-up reel 52. In the embodiment described herein, the conveyor is
plastic, but other materials may be employed as well.
It is to be understood that other forms of drive mechanisms, such as a
driven take-up reel, might be employed. It is also contemplated that the
conveyor could be an endless belt.
To induce the electrical surface charge on the conveyor 24, which causes
the tissue sections to adhere thereto, the conveyor 24 is passed between
two positively charged grids 54. The positively charged grids 54 are
positioned on opposing sides of the conveyor 24, and have a positive
charge maintained on their surface. Thus, as the conveyor 24 passes
between the positively charged surfaces, a positive electrostatic charge
is induced on the surface of the conveyor 24. After the conveyor 24 has a
positive electrostatic charge imposed thereon, it passes into the
dielectric oil 22. Since the oil 22 is a dielectric, the static charge
does not dissipate significantly therein, and, thus, a positive surface
charge is maintained on the surface of the conveyor 24.
While the embodiment of FIG. 1 depicts a positive surface charge imposed on
the surface of the conveyor 24, other methods of electrostatic adhesion
are equally well suited for such apparatus. For instance, a negative
surface charge can be imposed on the conveyor 24, or either negative or
positive surface charges can be imposed on the specimen 14 instead, or
opposite surface charges can be imposed on the conveyor 24 and specimen
14. Each of these provide the requisite electrostatic adhesion between the
tissue sections 26 and conveyor 24. Also, while charged grids are depicted
in the preferred embodiment, several alternate means for inducing
electrostatic charges on materials are equally well suited for carrying
out the present invention.
With continued reference to FIG. 1, the conveyor 24, having a surface
charge now imposed thereon, subsequently passes around a sprocketed wheel
40a which brings the conveyor 24 in close proximity with the stationary
blade 18 of the microtome 12. The influence of the surface charge imposed
on the surface of the conveyor 24 causes the tissue sections 26, and
associated tissue debris generated therewith, to migrate toward the
conveyor 24 and electrostatically adhere thereto. The tissue sectioning
and the initial electrostatic adhesion of the tissue sections 26 to the
conveyor 24 occur within the subzero dielectric oil 22, so that the tissue
sections 26 remain frozen, and, therefore, noninfectious.
Since the conveyor 24 is advanced in relation to the rate of tissue section
generation, as discussed above, each tissue section 26, and the tissue
debris generated with that particular section, occupies a separate space
along the length of the conveyor 24 separated from the other tissue
sections and their debris. Also, the electrostatic adhesion between the
tissue section 26 and the conveyor 24 prevents tissue and tissue debris
from becoming airborne after exiting the insulated tank 20.
As the conveyor 24 advances, the tissue sections 26 continue to adhere and
are advanced therewith. As shown in FIG. 1, the apparatus preferably
includes a headspace 55 in insulated tank 20 which may be filled with a
suitable gas, which may be a treatment gas, e.g., chlorine for
sterilizing, ozone for oxidizing or the like, or an inert gas such as
nitrogen. Preferably, the gas is maintained at a subzero temperature to
maintain the specimen and debris in a frozen state. It is appreciated that
several additional sprocketed wheels 40 may be employed to loop the
conveyor 24 one or more times within the headspace 55 so as to provide
additional reaction time therein.
Upon exiting the insulated tank 20, the tissue sections 26 are exposed to
radiation to sterilize any bacteria or viruses that may be present. As
shown in FIG. 1, the tissue sections 26 are passed between radiation
emitters 56. The radiation emitters 56 may be of any type well-known in
the art that is capable of providing the requisite radiation necessary to
sterilize the tissue sections 26. Alternatively, oxidizing lamps could be
employed in place of the radiation emitters 56 wherein the oxidizing lamps
generate ozone which sterilizes the tissue sections 26 while keeping the
chemistry and antigenicity of the tissue sections 26 intact. To assure
that the complete tissue section 26 is exposed to sufficient
sterilization, it is desirable to provide the radiation emitters 56 or
oxidizing lamps on either side of the conveyor 24 so that both sides of
the tissue section 26 are radiated directly. Any other suitable
sterilizing means may be employed as well.
After sterilization, the tissue sections are next prepared for viewing.
With reference to FIG. 1, it is seen that, upon exiting the radiation
emitters 56, the tissue specimens pass around the drive wheel 48
(discussed earlier) and then are passed into and out of staining and
fixing tanks 58. In practice, it is common for the tissue section to be
passed through a first staining tank 58 which contains a stabilizer to
inhibit cell decay, with the tissue sections 26 subsequently passed
through a plurality of additional staining tanks 58 depending upon the
tissue being examined. Whereas the illustrative embodiment of FIG. 1
depicts two staining tanks 58, as is well known in the art, any number of
staining tanks may be employed depending upon the analysis to be
performed.
Sprocketed wheels 40 are employed to move the conveyor 24, with the tissue
specimens 26 adhering thereto, into and out of the staining tanks 58, as
shown in FIG. 1. The number of sprocketed wheels 40 employed will vary
depending upon the number of staining tanks 58 employed.
After undergoing staining, the tissue sections 26 are transported to a
mounting station 60 at which the now sterilized and stained tissue
sections are transferred from the conveyor 24 to a glass slide 62. To
assure adhesion of the tissue sections 26 to the glass slide 62, a
methacrylate monomer 64 covers the surface of the glass slide 62. The
tissue section 26 is potted in the methacrylate monomer 64 which is then
polymerized by exposure to ultraviolet light emitted from an ultraviolet
light source 66. This solidifies the methacrylate monomer 64 with the
tissue section 26 therein such that the tissue section 26 is affixed to
the glass slide 62 for conventional viewing under a microscope 68.
Accordingly, tissue sections 26 can be taken from a tissue specimen 14 and
analyzed under a microscope 68 without an operator being exposed to
bacteria or viruses, and without tissue debris from one section
contaminating another section.
While the invention has been described with reference to a preferred
embodiment, it will be understood to those skilled in the art that various
changes may be made and equivalents may be substituted for elements
thereof without departing from the scope of the invention. In addition,
many modifications may be made to adapt a particular situation or material
to the teachings of the invention without departing from the essential
scope thereof. Therefore, it is intended that the invention not be limited
to the particular embodiment disclosed as the best mode contemplated for
carrying out this invention, but that the invention will include all
embodiments falling within the scope of the appended claims.
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